Method of the electrochemical detection of nucleic acid oligomer hybrids

ABSTRACT

The invention relates to a method for the electrochemical detection of sequence-specific nucleic acid oligomer hybridization events. To this end single DNA/RNA/PNA oligomer strands which at one end are covalently joined to a support surface and at the other, free end, covalently linked to a redox pair, are used as hybridization matrix (probe). As a result of treatment with the oligonucleotide solution (target) to be examined, the electric communication between the conductive support surface and the redox pair bridged by the single-strand oligonucleotide, which communication initially is either absent or very weak, is modified. In case of hybridization, the electric communication between the support surface and the redox pair, which is now bridged by a hybridized double-strand oligonucleotide, is increased. This permits the detection of a hybridization event by electrochemical methods such as cyclic voltametry, amperometry or conductivity measurement.

FIELD OF THE INVENTION

The present invention is directed to a modified nucleic acid oligomer,as well as a method of electrochemically detecting sequence-specificnucleic acid oligomer hybridization events.

BACKGROUND OF THE INVENTION

Generally, gel-electrophoretic methods with autoradiographical oroptical detection are used for DNA and RNA sequence analysis, e.g. indisease diagnosis, toxicological test procedures, genetic research anddevelopment, as well as in the agrarian and pharmaceutical sectors.

To illustrate the most significant gel-electrophoretic method withoptical detection (Sanger method), FIG. 1 b shows a DNA fragment withprimer. In the Sanger method, a DNA-containing solution is divided intofour samples and the primer of each sample is covalently modified with afluorescent dye that emits at a distinct wavelength. As illustrated inFIG. 1 b, deoxyribonucleoside triphosphate of bases A (adenine), T(thymine), C (cytosine), and G (guanine), i.e. dATP, dTTP, dCTP, anddGTP, are added to each sample to enzymatically replicate the singlestrand, starting at the primer, by means of DNA polymerase 1. Inaddition to the four deoxyribonucleoside triphosphates, each reactionmixture also contains sufficient 2′,3′-dideoxy analog (FIG. 1 a) of oneof these nucleoside triphosphates as a blocking base (one of each of thefour possible blocking bases per sample) to terminate replication at allpossible binding sites. After combining the four samples, all lengths ofreplicated DNA fragments having blocking-base-specific fluorescenceresult and can be gel-electrophoretically sorted according to length andcharacterized using fluorescent spectroscopy (FIG. 1 c).

Another optical detection method is based on the accumulation offluorescent dyes such as e.g. ethidium bromide on oligonucleotides. Thefluorescence of such dyes increases in comparison with the free solutionof the dye by about 20-fold when they accumulate on double-stranded DNAor RNA and can therefore be used to detect hybridized DNA or RNA.

In radiolabeling, ³²P is built into the phosphate skeleton of theoligonucleotides, with ³²P usually being added to the 5′-hydroxyl end bymeans of polynucleotide kinase. Thereafter, the labeled DNA ispreferably cleaved, under defined conditions, at one of each of the fournucleotide types, such that an average of one cleavage per chainresults. Thus, for a given base type, there are present in the reactionmixture chains extending from the ³²P-label to the position of that base(if there are multiple appearances of the base, chains of varyinglengths will result accordingly). The four fragment mixtures are thengel-electrophoretically separated on four lanes. Thereafter, anautoradiogram of the gel is prepared, from which the sequence can bedirectly read.

Some years ago, a further method of DNA sequencing was developed on thebasis of optical (or autoradiographical) detection, namely sequencing bymeans of oligomer hybridization (cf. e.g. Drmanac et al., Genomics 4,(1989), pp. 114–128 or Bains et al., Theor. Biol. 135, (1988), pp.303–307). In this method, a complete set of short oligonucleotides, oroligomers (probe oligonucleotides), e.g. all 65,536 possiblecombinations of the bases A, T, C, and G of an oligonucleotide octamerare bound to a support. The attachment occurs in an ordered gridconsisting of 65,536 test sites, with each larger amount of anoligonucleotide combination defining one test site, and the position ofeach individual test site (oligonucleotide combination) is known. Onsuch a hybridization matrix, the oligomer chip, a DNA fragment whosesequence is to be determined, the target, is labeled with fluorescentdye (or ³²P) and hybridized under conditions that allow only onespecific double-strand formation. In this way, the target DNA fragmentattaches only to the oligomers (in this example to the octamers) whosecomplementary sequence corresponds exactly to a portion (an octamer) ofits own sequence. Thus, all of the oligomer sequences (octamersequences) present in the fragment are determined by means of optical(or autoradiographical) detection of the binding position of thehybridized DNA fragment. Due to the overlapping of neighboring oligomersequences, the continuous sequence of the DNA fragment can be determinedusing suitable mathematical algorithms. The advantages of this methodlie in, among other things, the miniaturization of the sequencing andthus in the enormous amount of data that can be simultaneously capturedin one operation. In addition, primer and gel-electrophoretic separationof the DNA fragments can be dispensed with. This principle isdemonstrated by example in FIG. 2 for a 13-base-long DNA fragment.

The use of radioactive labels in DNA/RNA sequencing is associated withseveral disadvantages, such as e.g. elaborate, legally required safetyprecautions in dealing with radioactive materials, radiation, spatiallylimited resolution capacity (maximum 1 mm²) and sensitivity that is onlyhigh when the radiation of the radioactive fragments act on an X-rayfilm for an appropriately long time (hours to days). Although thespatial resolution can be increased by means of additional hardware andsoftware, and the detection time can be decreased by means ofβ-scanners, both of these involve considerable additional costs.

Some of the fluorescent dyes that are commonly used to label the DNA(e.g. ethidium bromide) are mutagenic and require appropriate safetyprecautions, as does the use of autoradiography. In nearly every case,the use of optical detection requires the use of one or more lasersystems, and thus experienced personnel and appropriate safetyprecautions. The actual detection of the fluorescence requiresadditional hardware such as e.g. optical components for amplificationand, in the case of varying stimulation and query wavelengths as in theSanger method, a control system. Thus, depending on the stimulationwavelengths required and the detection performance desired, considerableinvestment costs may result. In sequencing by means of hybridization onthe oligomer chip, detection is even more costly because, in addition tothe stimulation system, high-resolution CCD cameras (charge coupleddevice cameras) are needed for 2-dimensionally detecting the fluorescentspots.

Thus, although there are quantitative and extremely sensitive methodsfor DNA/RNA sequencing, these methods are time consuming, requireelaborate sample preparation and expensive equipment, and are generallynot available as portable systems.

DESCRIPTION OF THE INVENTION

Therefore, it is the object of the present invention to create fordetecting nucleic acid oligomer hybrids an apparatus and a method thatdo not exhibit the disadvantages of the state of the art.

According to the present invention, this object is solved by themodified oligonucleotide according to independent claim 1, by the methodof producing a modified oligonucleotide according to independent claims9 and 10, by the modified conductive surface according to independentclaim 11, the method of producing a modified conductive surfaceaccording to independent claim 21, and a method of electrochemicallydetecting oligomer hybridization events according to independent claim27.

The following abbreviations and terms are used herein:

Genetics DNA deoxyribonucleic acid RNA ribonucleic acid PNA peptidenucleic acid (Synthetic DNA or RNA in which the sugar-phosphate moietyis replaced by an amino acid. If the sugar-phosphate moiety is replacedby the —NH— (CH₂)₂—N(COCH₂-base)-CH₂CO— moiety, PNA will hybridize withDNA.) A adenine G guanine C cytosine T thymine base A, G, T, or C bpbase pair nucleic acid At least two covalently joined nucleotides or atleast two covalently joined pyrimidine (e.g. cytosine, thymine, oruracil) or purine bases (e.g. adenine or guanine). The term nucleic acidrefers to any backbone of the covalently joined pyrimidine or purinebases, such as e.g. to the sugar- phosphate backbone of DNA, cDNA, orRNA, to a peptide backbone of PNA, or to analogous structures (e.g. aphosphoramide, thiophosphate, or dithiophosphate backbone). Theessential feature of a nucleic acid according to the present inventionis that it can sequence-specifically bind naturally occurring cDNA orRNA. nucleic acid Nucleic acid of base length that is not furtherspecified (e.g. oligomer nucleic acid octamer: a nucleic acid having anybackbone in which 8 pyrimidine or purin bases are covalently bound toone another). oligomer Equivalent to nucleic acid oligomer. oligo-Equivalent to oligomer or nucleic acid oligomer, thus e.g. a nucleotideDNA, PNA, or RNA fragment of base length that is not further specified.oligo Abbreviation for oligonucleotide. dATP Deoxyribonucleosidetriphosphate of A (DNA moiety with the A base and two further phosphatesto build a longer DNA fragment or oligonucleotide). dGTPDeoxyribonucleoside triphosphate of G (DNA moiety with the G base andtwo further phosphates to build a longer DNA fragment oroligonucleotide). dCTP Deoxyribonucleoside triphosphate of C (DNA moietywith the C base and two further phosphates to build a longer DNAfragment or oligonucleotide). dTTP Deoxyribonucleoside triphosphate of T(DNA moiety with the T base and two further phosphates to build a longerDNA fragment or oligonucleotide). primer Initial complementary fragmentof an oligonucleotide, with the base length of the primer being onlyapprox. 4–8 bases. Serves as the starting point for enzymaticreplication of an oligonucleotide. mismatch To form the Watson Crickdouble-stranded oligonucleotide structure, the two single strandshybridize in such a way that the A (or C) base of one strand formshydrogen bonds with the T (or G) base of the other strand (in RNA, T isreplaced by uracil). Any other base pairing does not form hydrogenbonds, distorts the structure, and is referred to as a “mismatch.” dsdouble strand ss single strand Chemical Substances/Groups R Asubstituent or side chain of any organic residue not further specified.redox redox-active substance alkyl The term “alkyl” refers to asaturated hydrocarbon radical that is straight-chained or branched (e.g.ethyl, isopropyl, or 2,5-dimethylhexyl, etc.). When “alkyl” is used toindicate a linker or spacer, the term refers to a group having twoavailable valences for covalent linkage (e.g. —CH₂CH₂—, —CH₂CH₂CH₂—, or—CH₂C(CH₃)₂CH₂CH₂C(CH₃)₂CH₂—, etc.). Alkyl groups preferred assubstituents or side chains R are those of chain length 1–30 (longestcontinuous chain of atoms bound to one another). Alkyl groups preferredas linkers or spacers are those of chain length 1–20, especially ofchain length of 1–14, the chain length representing the shortestcontinuous link between linker or spacer-joined structures. alkenylAlkyl groups in which one or more of the C—C single bonds are replacedby C═C double bonds. alkinyl Alkyl or alkenyl groups in which one ormore of the C—C single or C═C double bonds are replaced by C≡C triplebonds. heteroalkyl Alkyl groups in which one or more of the C—H bonds orC—C single bonds are replaced by C—N, C═N, C—P, C═P, C—O, C═O, C—S, orC═S bonds. hetero- Alkenyl groups in which one or more C—H bonds, C—Calkenyl single, or C═C double bonds are replaced by C—N, C═N, C—P, C═P,C—O, C═O, C—S, or C═S bonds. heteroalkinyl Alkinyl groups in which oneor more of the C—H bonds, C—C single, C═C double, or C≡C triple bondsare replaced by C—N, C═N, C—P, C═P, C—O, C═O, C—S, or C═S bonds. linkerA molecular link between two molecules or between a surface atom,surface molecule, or surface molecule group and another molecule.Linkers can usually be purchased in the form of alkyl, alkenyl, alkinyl,heteroalkyl, heteroalkenyl, or heteroalkinyl chains, the chain beingderivatized in two places with (identical or different) reactive groups.These groups form a covalent chemical bond in simple/known chemicalreactions with the appropriate reaction partner. The reactive groups mayalso be photoactivatable, i.e. the reactive groups are activated only bylight of a specific or random wavelength. Preferred linkers are those ofchain length of 1–20, especially of chain length of 1–14, the chainlength representing here the shortest continuous link between thestructures to be joined, thus between the two molecules or between asurface atom, surface molecule, or surface molecule group and anothermolecule. spacer A linker that is covalently attached via the reactivegroups to one or both of the structures to be joined (see linker).Preferred spacers are those of chain length 1–20, especially of chainlength 1–14, the chain length representing the shortest continuous linkbetween the structures to be joined. (n × HS- A nucleic acid oligomer towhich n thiol functions are each spacer)-oligo attached via a spacer,where each spacer may have a different chain length (shortest continuouslink between the thiol function and the nucleic acid oligomer),especially any chain length between 1 and 14 each. These spacers, inturn, may be bound to various reactive groups that are naturally presenton the nucleic acid oligomer or that have been fixed thereto by means ofmodification, and “n” is any integer, especially a number between 1 and20. (n × R-S-S- A nucleic acid oligomer to which n disulfide functionsare spacer)-oligo each attached via a spacer, and any residue Rsaturates the disulfide function. Each spacer for attaching thedisulfide function to the nucleic acid oligomer may have a differentchain length (shortest continuous link between the disulfide functionand the nucleic acid oligomer), especially any chain length between 1and 14 each. These spacers, in turn, may be bound to various reactivegroups that are naturally present on the nucleic acid oligomer or thathave been fixed thereto by means of modification. The placeholder “n” isany integer, especially a number between 1 and 20. oligo-spacer- Twoidentical or different nucleic acid oligomers that are S-S-spacer-joined to each other via a disulfide bridge, the disulfide oligo bridgebeing attached to the nucleic acid oligomers via any two spacers and thetwo spacers potentially having differing chain lengths (shortestcontinuous link between the disulfide bridge and the respective nucleicacid oligomer), especially any chain length between 1 and 14 each, andthese spacers, in turn, potentially being bound to various reactivegroups that are naturally present on the nucleic acid oligomer or thathave been fixed thereto by means of modification. PQQ pyrroloquinolinequinone; corresponds to 4,5-dihydro-4,5-dioxo-1H-pyrrolo-[2,3-f]-quinoline-2,7,9-tricarboxylic acid) TEATFBtetraethylammonium-tetrafluoroborate sulfo-NHS N-hydroxysulfosuccinimideEDC (3-dimethylaminopropyl)-carbodiimide HEPESN-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] Tristrishydroxymethylamino methane EDTA ethylenediamine tetraacetate (sodiumsalt) cystamine (H₂N—CH₂—CH₂—S—)₂ Modified Surfaces/Electrodes micaMuskovite platelets, a support for the application of thin layers.Au-S-ss- Gold film on mica having a covalently applied monolayeroligo-PQQ of derivatized 12-bp single-strand oligonucleotide (sequence:TAGTCGGAAGCA) SEQ ID NO: 1. Here, the terminal phosphate group of theoligonucleotide at the 3′ end is esterified with (HO—(CH₂)₂—S)₂ to P—O—(CH₂)₂—S—S—(CH₂)₂—OH, homolytically cleaving the S—S bond and producingone Au-S-R bond each. The terminal thymine base at the 5′ end of theoligonucleotide is modified at the C-5 carbon with —CH═CH—CO—NH—CH₂—CH₂—NH₂ and the residue, in turn, is joined via its free amino groupwith a carboxylic-acid group of the PQQ by means of amidation. Au-S-ds-Au-S-ss-oligo-PQQ that is hybridized with the oligo-PQQ oligonucleotidecomplementary to the ss-oligo (sequence: TAGTCGGAAGCA SEQ ID NO: 1).Electrochemistry E The electrode potential on the working electrode. E₀Half-wave potential, the potential in the middle between the currentmaximums for oxidation and reduction of cyclic voltammetricallyreversible electrooxidation or reduction. i current density (current percm² of electrode surface) cyclic Recording a current-voltage curve. Thepotential of a voltammetry stationary working electrode is changedlinearly as a function of time, starting at a potential at which noelectrooxidation or reduction occurs, up to a potential at which aspecies that is solute or adsorbed on the electrode is oxidized orreduced (i.e. current flows). After running through the oxidation orreduction operation, which produces in the current-voltage curve aninitially increasing current and, after reaching a maximum, a graduallydecreasing current, the direction of the potential feed is reversed. Thebehavior of the products of electrooxidation or electroreduction is thenrecorded in reverse run. amperometry Recording a current-time curve.Here, the potential of a stationary working electrode is set, e.g. bymeans of a potential jump, to a potential at which the electrooxidationor reduction of a solute or adsorbed species occurs, and the flowingcurrent is recorded as a function of time.

The present invention is directed to a nucleic acid oligomer that ismodified by the chemical attachment of a redox-active substance.According to the present invention, the nucleic acid oligomer is acompound consisting of at least two covalently joined nucleotides or atleast two covalently joined pyrimidine (e.g. cytosine, thymine, oruracil) or purine bases (e.g. adenine or guanine), preferably a DNA,RNA, or PNA fragment. As used herein, the term nucleic acid refers toany backbone of the covalently joined pyrimidine or purine bases, suchas e.g. to the sugar-phosphate backbone of DNA, cDNA, or RNA, to apeptide backbone of PNA, or to analogous backbone structures such ase.g. a thiophosphate, a dithiophosphate, or a phosphoramide backbone.The essential feature of a nucleic acid according to the presentinvention is that it can sequence-specifically bind naturally occurringcDNA or RNA. The terms “(probe) oligonucleotide,” “nucleic acid,” and“oligomer” are used as alternatives to the term “nucleic acid oligomer.”

The redox-active substance is selectively oxidizable and reducible at apotential φ, where φ satisfies the condition 2.0 V≧φ≧−2.0 V. Thepotential refers here to the free, unmodified, redox-active substance ina suitable solvent, measured against normal hydrogen electrode.According to the present invention, the potential range 1.7 V≧φ≧−1.7 Vis preferred, the range 1.4 V≧φ≧−1.2 V being particularly preferred, andthe range 0.9 V≧φ≧−0.7 V, in which the redox-active substances of theapplication example are reduced and reoxidized, being most particularlypreferred. In addition, the present invention is directed to aconductive surface to which a nucleic acid oligomer having an attachedredox-active substance is chemically bound, directly or indirectly (viaa spacer). Furthermore, the present invention is directed to a method ofproducing a modified conductive surface, wherein a modified nucleic acidoligomer is applied to a conductive surface. According to a furtheraspect, the present invention is directed to a method that allowselectrochemical detection of molecular structures, especiallyelectrochemical detection of DNA/RNA/PNA fragments in a probe solutionby means of sequence-specific nucleic acid oligomer hybridization.Detection of hybridization events by means of electrical signals is asimple and economical method and, in a battery-operated variation of asequencing device, allows on-site application.

Binding a Redoxactive Moiety to a Nucleic Acid Oligomer

For the method of the present invention, it is necessary to bind aredox-active substance to a nucleic acid oligomer. According to thepresent invention, any redox-active substance may be used for thispurpose as long as it is selectively oxidizable and reducible at apotential φ that satisfies the condition 2.0 V≧φ≧−2.0 V. The potentialrefers here to the free, unmodified, redox-active substance in asuitable solvent, measured against normal hydrogen electrode. Accordingto the present invention, the potential range 1.7 V≧φ≧−1.7 V ispreferred, the range 1.4 V≧φ≧−1.2 V is particularly preferred, and therange 0.9 V≧φ≧−0.7 V, in which the redox-active substances of theapplication example are reduced and reoxidized, is most particularlypreferred. According to the present invention, the term “selectivelyoxidizable and reducible” is understood to mean a redox reaction, i.e.giving up or taking in an electron, that occurs selectively at thelocation of the redox-active substance. Thus, in the end, no other partof the nucleic acid oligomer is reduced or oxidized by the potentialapplied, but rather, exclusively the redox-active substance bound to thenucleic acid oligomer.

According to the present invention, a redox-active substance isunderstood to mean any molecule that, in the electrochemicallyaccessible potential range of the respective support surface(electrode), can be electrooxidized/electroreduced by applying anexternal voltage to that electrode. In additon to common organic andanorganic redox-active substances such as e.g. hexacyanoferrates,ferrocenes, acridines, or phtalocyanines, redox-active dyes such as e.g.(metallo-) porphyrins of the general Formula 1, (metallo-) chlorophyllsof the general Formula 2, or (metallo-) bacteriochlorophylls of thegeneral Formula 3, (colored) naturally occurring oxidation agents suchas e.g. flavines of the general Formula 4, pyridine-nucleotides of thegeneral Formula 5 or pyrrolo-quinoline quinones (PQQ) of the generalFormula 6, or other quinones such as e.g. 1,4-benzoquinones of thegeneral Formula 7, 1,2-benzoquinones of the general Formula8,1,4-naphthoquinones of the general Formula 9,1,2-naphthoquinones ofthe general Formula 10, or 9,10-anthraquinones of the general Formula 11are particularly suitable for attachment to the probe oligonucleotide.

M=2H, Mg, Zn, Cu, Ni, Pd, Co, Cd, Mn, Fe, Sn, Pt, etc.; R₁ to R₁₂ are,independently of one another, H or any alkyl, alkenyl, alkinyl,heteroalkyl, heteroalkenyl, or heteroalkinyl substituents.

R₁ to R₈ are, independently of one another, H or any alkyl, alkenyl,alkinyl, heteroalkyl, heteroalkenyl, or heteroalkinyl substituents.

According to the present invention, a redox-active substance iscovalently bound to an oligonucleotide by means of the oligonucleotidereacting with the redox-active substance. This bond can be achieved inthree different ways:

a) The reactive group for forming a bond at the nucleic acid oligomer isa free phosphoric-acid, sugar-C-3-hydroxy, carboxylic-acid, or aminegroup of the oligonucleotide backbone, especially a group at one of thetwo ends of the oligonucleotide backbone. The free, terminalphosphoric-acid, sugar-C-3-hydroxy, carboxylic-acid, or amine groupsexhibit increased reactivity and thus easily undergo typical reactionssuch as e.g. amidation with (primary or secondary) amino groups or withacid groups, esterification with (primary, secondary, or tertiary)alcohols or with acid groups, thioester formation with (primary,secondary, or tertiary) thioalcohols or with acid groups, orcondensation of amine and aldehyde with subsequent reduction of theresultant CH═N bond to a CH₂—NH bond. The coupling group required forthe covalent attachment of the redox-active substance (acid, amine,alcohol, thioalcohol, or aldehyde function) is either naturally presenton the redox-active substance or is obtained by means of chemicalmodification of the redox-active substance.

b) The nucleic acid oligomer is modified with a reactive group at theoligonucleotide backbone or at a base via a covalently attachedmolecular moiety (spacer) of any composition and chain length(representing the shortest continuous link between the structures to bejoined), especially of chain length 1 to 14. The modification preferablyoccurs at one of the ends of the oligonucleotide backbone or at aterminal base. Spacers may be e.g. an alkyl, alkenyl, alkinyl,heteroalkyl, heteroalkenyl, or heteroalkinyl substituent. Possiblesimple reactions for forming the covalent bond between the redox-activesubstance and the nucleic acid oligomer so modified are, as describedunder a), amidation from an acid and amino group, esterification from anacid and alcohol group, thioester formation from an acid and thioalcoholgroup, or condensation of aldehyde and amine with subsequent reductionof the resultant CH═N bond to a CH₂—NH bond.

According to a preferred embodiment, the nucleic acid oligomer ismodified using a redox-active substance that exhibits regions having apredominantly planar p-π-orbital system extended in a plane, such ase.g. the PQQ of Example 1 or the quinones of Formulas 5 or 7–12 or theporphinoid structures of Formulas 1–4 or the pyridine nucleotides of thegeneral Formula 6, or derivatives of these redox-active substances. Inthis case, the spacer via which the redox-active substance is bound tothe nucleic acid oligomer can be selected in such a way that the planeof the p-π-orbitals of the redox-active substance can arrange itselfparallel to the p-π-orbitals of the nucleic acid oligomer bases thatborder on the redox-active substance. This spatial arrangement of theredox-active substance with partially planar p-π-orbitals extended in aplane proves to be particularly favorable.

c) In synthesizing the nucleic acid oligomer, a terminal base will bereplaced by the redox-active substance.

According to the present invention, binding the redox-active substanceto the oligonucleotide as described under a) and b) may occur before orafter binding the oligonucleotide to the conductive surface. Theattachment of the redox-active substance to the oligonucleotide bound tothe conductive surface then likewise occurs as described under a) andb).

If there are several different oligonucleotide combinations (test sites)on a common surface, it is advantageous to standardize the (covalent)attachment of the redox-active substance to the probe oligonucleotidesfor the entire surface by the appropriate choice of reactive group atthe free probe oligonucleotide ends of the various test sites.

The Conductive Surface

According to the present invention, the term “conductive surface” refersto any support having an electrically conductive surface of anythickness, especially surfaces made of platinum, palladium, gold,cadmium, mercury, nickel, zinc, carbon, silver, copper, iron, lead,aluminum, and manganese. According to the present invention, the terms“electrode” and “conductive (support) surface” are used as alternativesto “conductive surface.”

In addition, any doped or non-doped semiconductor surfaces of anythickness may also be used. All semiconductors are useful in the form ofpure substances or as mixtures. Examples include, but are not limitedto, carbon, silicon, germanium, α tin, and Cu(I) and Ag(I) halides ofany crystal structure. Likewise suitable are all binary compounds of anycomposition and any structure of the elements of groups 14 and 16, ofthe elements of groups 13 and 15, and of the elements of groups 15 and16. In addition, ternary compounds of any composition and any structureof the elements of groups 11, 13, and 16 or of the elements of groups12, 13, and 16 may be used. The designations of the groups of theperiodic system refer to the IUPAC recommendation of 1985.

Binding an Oligonucleotide to the Conductive Surface

According to the present invention, a nucleotide is linked directly orvia a linker/spacer with the support surface atoms or molecules of aconductive support surface of the kind described above. This bond may becarried out in three different ways:

a) The surface is modified in such a way that a reactive molecule groupis accessible. This may occur by means of direct derivatization of thesurface molecules, e.g. by means of wet chemical or electrochemicaloxidation/reduction. Thus e.g. the surface of graphite electrodes can bewet-chemically supplied with aldehyde or carboxylic-acid groups by meansof oxidation. Electrochemically, it is possible e.g. by means ofreduction in the presence of aryl-diazonium salts to couple thecorresponding (functionalized, i.e. supplied with a reactive group) arylradical, or by means of oxidation in the presence of R′CO₂H to couplethe (functionalized) R′ radical to the graphite electrode surface. Anexample of direct modification of semiconductor surfaces is thederivatization of silicon surfaces to reactive silanols, i.e. siliconsupports having Si—OR″ groups on the surface, where both R″ and R′ areany functionalized organic residue (e.g. alkyl, alkenyl, alkinyl,heteroalkyl, heteroalkenyl, or heteroalkinyl substituent).Alternatively, the entire surface may be modified by covalentlyattaching a reactive group of a bifunctional linker such that amonomolecular layer consisting of any molecules and containing areactive group, preferably terminally, results on the surface. The term“bifunctional linker” is understood to mean any molecule of any chainlength, especially of chain lengths 2-14, having two identical(homobifunctional) or two different (heterobifunctional) reactivemolecule groups.

If several different test sites are to be formed on the surface bymaking use of the methodology of photolithography, then at least one ofthe reactive groups of the homobifunctional or heterobifunctionallinkers is a photoinducible reactive group, i.e. a group that becomesreactive only upon irradiation with light of a specific or randomwavelength. This linker is applied in such a way that the/aphotoactivatable reactive group is available after the linker iscovalently attached to the surface. The nucleic acid oligomers arecovalently attached to the surface so modified and are, themselves,modified with a reactive group, preferably near an end of the nucleicacid oligomer, via a spacer of any composition and chain length,especially of chain length 1–14. The reactive group of theoligonucleotide is a group that reacts directly (or indirectly) with themodified surface to form a covalent bond. In addition, a furtherreactive group may be bound to the nucleic acid oligomers near itssecond end, this reactive group, in turn, being attached, as describedabove, directly or via a spacer of any composition and chain length,especially of chain length 1–14. Furthermore, as an alternative to thisfurther reactive group, the redox-active substance may be attached atthis second end of the nucleic acid oligomer.

b) The nucleic acid oligomer to be applied to the conductive surface ismodified with one or more reactive groups via a covalently attachedspacer of any composition and chain length, especially of chain length1–14, these reactive groups being located preferably near an end of thenucleic acid oligomer. The reactive groups are groups that can reactdirectly with the unmodified surface. Some examples are: (i) thiol-(HS)or disulfide-(S—S—) derivatized nucleic acid oligomer of the generalformula (n×HS-spacer)-oligo, (n×R—S—S-spacer)-oligo, oroligo-spacer-S—S-spacer-oligo that react with a gold surface to formgold-sulfur bonds, or (ii) amines that accumulate on platinum or siliconsufaces by means of chemisorption or physisorption. In addition, afurther reactive group may be bound to the nucleic acid oligomers nearits second end, this reactive group, in turn, being attached, asdescribed above, directly or via a spacer of any composition and chainlength, especially of chain length 1–14. Furthermore, as an alternativeto this further reactive group, the redox-active substance may beattached at this second end of the oligonucleotide. Particularly nucleicacid oligomers that are modified with several spacer-bridged thiol ordisulfide bridges ((n×HS-spacer)-oligo or (n×R—S—S-spacer)-oligo) havethe advantage that such nucleic acid oligomers can be applied to theconductive surface at a particular setting angle (angle between thesurface normal and the helix axis of a double-stranded helical nucleicacid oligomer or between the surface normal and the axis perpendicularto the base pairs of a double-stranded non-helical nucleic acidoligomer) if the spacers attaching the thiol or disulfide functions tothe nucleic acid oligomer possess an increasing or decreasing chainlength as viewed from an end of the nucleic acid.

c) Phosphoric-acid, sugar-C-3-hydroxy, carboxylic-acid, or amine groupsof the oligonucleotide backbone, especially terminal groups, are used asthe reactive group on the probe nucleic acid oligomer. Thephosphoric-acid, sugar-C-3-hydroxy, carboxylic-acid, or amine groupsexhibit greater reactivity and thus easily undergo typical reactionssuch as amidation with (primary or secondary) amino or acid groups,esterification with (primary, secondary, or tertiary) alcohols or acidgroups, thioester formation with (primary, secondary, or tertiary)thioalcohols or acid groups, or condensation of amine and aldehyde withsubsequent reduction of the resultant CH═N bond to a CH₂—NH bond. Inthis case, the coupling group required for the covalent attachment tothe phosphoric-acid, sugar-C-3-hydroxy, carboxylic-acid, or amine groupis part of the surface derivatization with a (monomolecular) layer ofany molecule length, as described under a) in this section, or thephosphoric-acid, sugar-C-3-hydroxy, carboxylic-acid, or amine group canreact directly with the unmodified surface, as described under b) inthis section. In addition, a further reactive group may be bound to theoligonucleotides near its second end, this reactive group, in turn,being attached, as described above, directly or via a spacer of anycomposition and chain length, especially of chain length 1–14.Furthermore, as an alternative to this further reactive group, theredox-active substance may be attached at this second end of the nucleicacid oligomer.

Alternatively, binding the oligonucleotide to the conductive surface mayoccur before or after attaching the redox-active substance to theoligonucleotide, or before or after attaching the spacer supplied with areactive group for binding the redox-active substance. Binding thealready modified oligonucleotide to the conductive surface, i.e. bindingto the surface after attaching the redox-active substance to theoligonucleotide, or after attaching the spacer supplied with a reactivegroup for binding the redox-active substance, likewise takes place asdescribed under a) to c) (in the section “Binding an Oligonucleotide tothe Conductive Surface”).

In producing the test sites, care must be taken when attaching thesingle-strand oligonucleotides to the surface that a sufficiently largedistance remains between the individual oligonucleotides to provide thenecessary space for a hybridization with the target oligonucleotide. Tothis end, two different methods of proceeding, among others, presentthemselves:

1.) Producing a modified support surface by attaching a hybridizedoligonucleotide, i.e. a support surface derivatization with hybridizedprobe oligonucleotide instead of with single-strand probeoligonucleotide. The oligonucleotide strand used for hybridization isunmodified (the surface attachment is carried out as described undera)–c) in the section “Binding an Oligonucleotide to the ConductiveSurface”). Thereafter, the hybridized oligonucleotide double strand isthermally dehybridized, thus producing asingle-strand-oligonucleotide-modified support surface having greaterdistance between the probe oligonucleotides.2.) Producing a modified support surface by attaching a single-strand ordouble-strand oligonucleotide, and adding during support surfacederivatization a suitable monofunctional linker that, in addition to thesingle-strand or double-strand oligonucleotide, is also bound to thesurface (the surface attachment is carried out as described under a)–c)in the section “Binding an Oligonucleotide to the Conductive Surface”).According to the present invention, the monofunctional linker has achain length that is identical to the chain length of the spacer betweenthe support surface and the oligonucleotide, or that differs by amaximum of eight chain atoms. If double-strand oligonucleotide is usedfor support-surface derivatization, the hybridized oligonucleotidedouble strand is thermally dehybridized after attaching thedouble-strand oligonucleotide and the linker to the support surface, asdescribed under 1.) above. By simultaneously attaching a linker to thesurface, the distance between the single-strand or double-strand nucleicacid oligomers that are likewise bound to the surface is increased. If adouble-strand nucleic acid oligomer is used, this effect is amplifiedfurther by the subsequent thermal dehybridization.Method of Electrochemically Detecting Nucleic Acid Oligomer Hybrids

Advantageously, according to the method of electrochemical detection,several probe oligonucleotides varying in sequence, ideally allnecessary combinations of the nucleic acid oligomer, are applied to anoligomer chip or DNA chip to reliably detect the sequence of any targetoligomer or of a (fragmented) target DNA, or to seek andsequence-specifically detect mutations in the target. To this end, thesupport surface atoms or molecules of a defined area (a test site) arelinked with DNA/RNA/PNA oligonucleotides of known but random sequence ona conductive support surface, as described above. In a most generalembodiment, however, the DNA chip may also be derivated with a singleprobe oligonucleotide. Preferred probe oligonucleotides are nucleic acidoligomers (DNA, RNA, or PNA fragments) of base length 3 to 50,preferably of length 5 to 30, particularly preferably of length of 7 to25. According to the present invention, a redox-active substance isbound to the probe oligonucleotides either before or after the latter isbound to the conductive surface.

If the modification of the probe oligonucleotides occurs before the bondto the conductive surface, then the already modifed probeoligonucleotides are bound to the conductive surface as described above.Alternatively, the non-modified probe oligonucleotides bound to theconductive surface are modified with a redox-active substance at thesecond, free end of the oligonucleotide chain, directly or indirectlyvia a spacer.

In both cases, a surface hybrid of the general structureelec-spacer-ss-oligo-spacer-redox (FIG. 3) results. The electricalcommunication between the (conductive) support surface and theredox-active substance (“redox”) bridged via a single-strandoligonucleotide in the general structureelec-spacer-ss-oligo-spacer-redox is weak or not present at all. Thebridges may, of course, also be carried out without spacers or with onlyone spacer (elec-ss-oligo-spacer-redox or elec-spacer-ss-oligo-redox).In a next step, the test sites are brought into contact with theoligonucleotide solution (target) to be examined. Hybridization willonly occur if the solution contains oligonucleotide strands that arecomplementary to the probe oligonucleotides bound to the conductivesurface, or at least widely complementary. In the case of hybridizationbetween the probe and target oligonucleotide, there will be increasedconductivity between the support surface and the redox-active substancebecause these are now bridged via the oligonucleotide composed of adouble strand (shown schematically in FIG. 3 using an example of theelec-spacer-ss-oligo-spacer-redox).

Because of the change in the electrical communication between the(conductive) support surface and the redox-active substance due to thehybridization of the probe oligonucleotide and the oligonucleotidestrand (target) complementary to it, a sequence-specific hybridizationevent can thus be detected using electrochemical methods such as e.g.cyclic voltammetry, amperometry, or conductivity measurements.

In a particularly preferred embodiment of the present invention, aredox-active substance is used that exhibits regions having apredominantly planar p-π-orbital system extended in a plane, such ase.g. the PQQ of Example 1 (cf. FIG. 3), or the quinones of Formula 5 or7–12 or the porphinoid structures of Formulas 1–4, the pyridinenucleotides of the general Formula 6 and derivatives of theseredox-active substances. In this case, the spacer between the nucleicacid oligomer and the redox-active substance is selected in such a waythat the plane of the p-π-orbitals of the redox-active substance canarrange itself parallel to the p-π-orbitals of the base pair of thenucleic acid oligomer hybridized with the complimentary strand andbordering on the redox-active substance. This spatial arrangement of theredox-active substance with partially planar p-7α-orbitals extended in aplane proves to be particularly favorable for the electricalconductivity of the double-strand nucleic acid oligomers.

In cyclic voltammetry, the potential of a stationary working electrodeis changed linearly as a function of time. Starting at a potential atwhich no electrooxidation or reduction occurs, the potential is changeduntil the redox-active substance is oxidized or reduced (i.e. currentflows). After running through the oxidation or reduction operation,which produces in the current-voltage curve an initially increasingcurrent, a maximum current (peak), and then a gradually decreasingcurrent, the direction of the potential feed is reversed. The behaviorof the products of electrooxidation or electroreduction is then recordedin reverse run.

An alternative electrical detection method, amperometry, is madepossible by the fact that the redox-active substance is electrooxidized(electroreduced) by applying a suitable, constant electrode potential,but rereducing (reoxidizing) the redox-active substance to its originalstate is achieved, not by changing the electrode potential as in cyclicvoltammetry, but rather by means of a suitable reducing agent (oxidizingagent) added to the target solution, closing the current circuit of theentire system. As long as reducing agent (oxidizing agent) is present,or as long as the consumed reducing agent (oxidizing agent) is rereduced(reoxidized) on the counter electrode, current flows that can beamperometrically detected and that is proportional to the number ofhybridization events.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be explained in greater detail using the followingapplication example and the accompanying drawings.

FIG. 1 Shows a schematic illustration of the Sanger method ofoligonucleotide sequencing; FIG. 2 Shows a schematic illustration ofoligonucleotide sequencing by means of hybridization on a chip; FIG. 3Shows a schematic illustration of the surface hybrid of the generalstructure elec-spacer-ss-oligo-spacer-redox with a 12-bp probeoligonucleotide of the exemplary sequence 5′- TAGTCGGAAGCA-3′ SEQ ID NO:1 (left) and Au-S-ss-oligo- PQQ in the hybridized state as an embodimentexample of an elec-spacer-ss-oligo-spacer-redox; only a portion of theprobe oligonucleotide having a hybridized complementary strand is shown(right), the attachment of the oligonucleotide to the surfaceredox-active substance PQQ occurred via the spacer—CH₂—CH═CH—CO—NH—CH₂—CH₂—NH—; FIG. 4 Shows a cyclic voltammogram of atest site consisting of Au-S- ss-oligo-PQQ (dotted) compared with anidentical test site with completely hybridized target(Au-S-ds-oligo-PQQ, solid line); FIG. 5 Shows a cyclic voltammogram of atest site with completely hybridized target (Au-S-ds-oligo-PQQ) (solidline) compared with a test site with hybridized target that exhibits 2base-pair mismatches (Au-S-ds-oligo-PQQ with 2 bp mismatches, broken)

CARRYING OUT THE INVENTION

An exemplary test site with hybridized target (Au—S-ds-oligo-PQQ) of thegeneral structure elec-spacer-ds-oligo-spacer-redox is shown in FIG. 3.In the example of FIG. 3, the support surface is a gold electrode. Thelink between the gold electrode and the probe oligonucleotide was formedwith the linker (HO—(CH₂)₂—S)₂, which was esterified with the terminalphosphate group at the 3′ end to P—O—(CH₂)₂—S—S—(CH₂)₂—OH and, followinghomolytic cleavage of the S—S bond at the gold surface, produced oneAu—S bond each, with which 2-hydroxy-mercaptoethanol andmercaptoethanol-bridged oligonucleotide was coadsorbed on the surface.The redox-active substance in the example of FIG. 3 is tricarboxylicpyrrolo-quinoline quinone (PQQ) and one of the three carboxylic acidfunctions of the PQQ (in the example, the C-7—CO₂H function) was used tocovalently attach the PQQ to the probe oligonucleotide (amidation anddehydration with the terminal amino function of the—CH═CH—CO—NH—CH₂—CH₂—NH₂ spacer attached to the C-5 position of the 5′thymine). Both free, unmodified PQQ and PQQ bridged with the supportsurface via a short spacer of chain length 1–6, such as e.g.—S—(CH₂)₂—NH—, or via (modified) double-strand oligonucleotide, e.g. inHEPES buffer with 0.7 molar addition of TEATFB (see abbreviations), isselectively reduced and oxidized in the potential range 0.7 V≧φ≧0.0 V,measured against normal hydrogen electrode.

The electrical communication between the (conductive) support surfaceand the redox pair bridged via single-strand oligonucleotide in thegeneral structure elec-spacer-ss-oligo-spacer-redox is weak or notpresent at all. For the exemplary test site Au—S-ss-oligo-PQQ (with12-bp probe oligonucleotides), this is shown with cyclic voltammetry(FIG. 4). Without wanting to be bound to a theoretical description, itis assumed that the negative charges of the phosphate skeleton cause amutual repulsion of the oligonucleotide single strands and thus force aformation of the -spacer-ds-oligo-spacer-redox chain (in the directionof the helix axis) at an angle +<70° to the normal of the supportsupport (“standing tubes”). The (hybridized) test site Au—S-ds-oligo-PQQof FIG. 3 exhibits a formation having φ=30°. Due to the length of the-spacer-ds-oligo-spacer-redox chain (e.g. approx. 40 Å length of a12-base-pair oligonucleotide; the spacers and the attached PQQ are about10 Å long), if φ<70°, a distance of >17 Å results between the surfacesupport and the redox-active substance. As a result, the possibility ofa direct electron or hole transfer between the support and theredox-active substance can be excluded. By treating the test site(s)with an oligonucleotide solution to be examined, in the case ofhybridization between probe and target, there will be increasedconductivity between the support surface and the redox pair bridged viaa double-strand oligonucleotide. The change in the conductivitymanifests itself cyclic voltammetrically in a significant current flowbetween the support surface and the redox-active substance (FIG. 4). Itis thus possible to detect the sequence-specific hybridization of thetarget with the probe oligonucleotides using electrochemical methodssuch as e.g. cyclic voltammetry.

In addition, defective base pairings (base-pair mismatches) can berecognized by means of a modified cyclic voltammetric characteristic(FIG. 5). A mismatch manifests itself in a greater potential differencebetween the current maximums of electroreduction and electroreoxidation(reversal of electroreduction when potential feed direction isreversed), or electrooxidation and electrorereduction in a cyclicvoltammetrically reversible electron transfer process between theelectrically conducting support surface and the redox-active substance.This fact has an advantageous effect primarily on amperometric detectionbecause there, the current can be tested at a potential at which theperfectly hybridizing oligonucleotide target supplies significantcurrent, but the defectively paired oligonucleotide target does not. Inthe example of FIG. 5, this is possible at a potential E–E₀ of approx.0.03 V.

EXAMPLE 1

Producing the Au—S-ds-oligo-PQQ oligonucleotide electrode. Theproduction of Au—S-ds-oligo-PQQ is divided into 4 subsections, namelyproducing the support surface, hybridizing the probe oligonucleotidewith the complementary double strand (hybridization step), derivatizingthe support surface with the double-strand oligonucleotide (incubationstep) and attaching the redox-active substance (redox step).

An approx. 100 nm thin gold film on mica (muscovite platelets) forms thesupport for the covalent attachment of the double-strandoligonucleotides. To this end, freshly cleaved mica was purified with anargon-ion plasma in an electrical discharge chamber and gold (99.99%)was applied, by means of electrical discharge, in a layer thickness ofapprox. 100 nm. Thereafter, the gold film was freed of surfaceimpurities (oxidation of organic accumulations) with 30% H₂O₂, /70%H₂SO₄ and immersed in ethanol for approx. 20 minutes to dispel anyoxygen adsorbed to the surface. After rinsing the support surface withbidistilled water, a previously prepared 1×10⁴ molar solution of the(modified) double-strand oligonucleotide is applied to the horizontallydisposed surface, such that the entire support surface is moistened(incubation step, see also below).

To prepare the ds oligonucleotide solution, a double-modified 12-bpsingle-strand oligonucleotide of the sequence 5′-TAGTCGGAAGCA-3′ SEQ IDNO: 1 was used, which is esterified with (HO—(CH₂)₂—S)₂ at the phosphategroup of the 3′ end to P—O—(CH₂)₂—S—S—(CH₂)₂—OH. At the 5′ end, theterminal base of the oligonucleotide, thymine, is modified at the C-5carbon with —CH═CH—CO—NH—CH₂—CH₂—NH₂. A 2×10⁴ molar solution of thisoligonucleotide in the hybridization buffer (10 mM Tris, 1 mM EDTA, pH7.5 with 0.7 molar addition of TEATFB, see abbreviations) was hybridizedwith a 2×10⁻⁴ molar solution of the (unmodified) complementary strand inthe hybridization buffer at room temperature for approx. 2 hours(hybridization step). During a reaction time of approx. 12–24 h, thedisulfide spacer P—O—(CH₂)₂—S—S—(CH₂)₂—OH of the oligonucleotide washomolytically cleaved. In this process, the spacer forms a covalent Au—Sbond with the Au atoms of the surface, thus causing to a 1:1coadsorption of the ds-oligonucleotide and the2-hydroxy-mercaptoethanol.

The gold electrode modified in this way with a dense (1:1) monolayerconsisting of ds-oligonucleotide and 2-hydroxy-mercaptoethanol waswashed with bidistilled water and subsequently moistened with a solutionof 3×10³ molar quinone PQQ, 102 molar EDC, and 10⁻² molar sulfo-NHS inHEPES buffer. After a reaction time of approx. 1 h, the—CH═CH—CO—NH—CH₂—CH₂—NH₂ spacer covalently attaches the PQQ (amidationbetween the amino group of the spacer and an acid function of the PQQ,redox step).

Resolution of the surface composition with XPS (X-Ray PhotoelectronSpectroscopy) showed a maximally densely packed monolayer of 1:1coadsorbed ds-oligonucleotide and 2-hydroxy-mercaptoethanol (4.7×10¹²ds-oligonucleotide/cm²), the long axis (direction of the helix axis) ofthe ds-oligonucleotides forming an angle of φ≈ 300 with the surfacenormal of the gold surface.

EXAMPLE 2

Producing the Au—S-ss-oligo-PQQ oligonucleotide electrode. Analogouslyto the production of the Au—S-ds-oligo-PQQ system, the support surfaceis derivatized with modified single-strand oligonucleotide, dispensingwith only the hybridization of the modified oligonucleotide of thesequence 5′-TAGTCGGAAGCA-3′ SEQ ID NO: 1 with its complementary strandand, in the incubation step, using only the double-modified 12 bpsingle-strand probe oligonucleotide (see Example 1) in the form of a1×10⁻⁴ molar solution in water and in the presence of 10⁻² molar Tris,10⁻³ molar EDTA and 0.7 molar TEATFB (or 1 molar NaCl) at pH 7.5. Theredox step was carried out as indicated in Example 1.

EXAMPLE 3

Producing the Au—S-ds-oligo-PQQ oligonucleotide electrode having 2 bpmismatches. The production of a support surface derivatized withmodified double-strand oligonucleotide was carried out analogously tothe production of the Au—S-ds-oligo-PQQ system, but only in hybridizingthe modified oligonucleotide of the sequence 5′-TAGTCGGAAGCA-3′ SEQ IDNO: 1 was a complementary strand used (sequence: 5′-ATCAGATTTCGT-3′) SEQID NO: 2, in which bases no. 6 and 7 (counted from the 5′ end), whichare actually complementary, were modified from C to A or from C to T tointroduce two base-pair mismatches.

EXAMPLE 4

Producing an Au—S-ss-oligo-PQQ oligonucleotide electrode having greaterinter-oligonucleotide distance. In producing the test sites, care mustbe taken that, in derivatizing the support surface with single-strandprobe oligonucleotide, sufficient space remains between the attachedsingle-strands to allow a hybridization with the target oligonucleotide.To this end, three different methods of proceeding present themselves:(a) Producing an Au—S-ds-oligo-PQQ electrode as described in Example 1,with subsequent thermal dehybridization of the double strands attemperatures of T>40° C. (b) Producing an Au-ss-oligo-PQQ electrode asdescribed in Example 2, but in the incubation step for derivatizing thegold surface with (double-derivatized) single-strand oligonucleotide,10⁻⁵ to 10⁻¹ molar 2-hydroxy-mercaptoethanol or another thiol ordisulfide linker of suitable chain length is added (depending on thedesired inter-oligonucleotide distance) and coadsorbed on the goldsurface together with the single-strand oligonucleotide. (c) Producingan Au-ss-oligo-PQQ electrode as described in Example 2, but omitting the0.7 molar addition of electrolytes (TEATFB in the Example) in theincubation step for derivatizing the gold surface with(double-derivatized) single-strand oligonucleotide. Due to the absenceof the salt, the phosphate groups and nitrogen-base atoms of theoligonucleotide are not electrostatically shielded and interact stronglywith the gold surface. Because of this, a shallow accumulation of theoligonucleotides results on the electrode surface (φ>600) andsignificantly fewer oligonucleotides are bound per surface unit.Thereafter, the oligonucleotides may be returned to the desired positionby covalently attaching in a second incubation step (before or afterattaching the PQQ) a 2-hydroxy-mercaptoethanol or another thiol ordisulfide linker of suitable chain length to the surface gold atoms thatare still free. To do this, the electrode that is less densely coveredwith single-strand oligonucleotide is moistened, before or aftermodification with PQQ (Au—S-ss-oligo or Au—S-ss-oligo-PQQ), with anapprox. 5×10⁻² molar solution of 2-hydroxy-mercaptoethanol or anotherthiol or disulfide linker of suitable chain length in ethanol or HEPESbuffer (or a mixture thereof, depending on the solubility of the thiol),and incubated for 2–24 h.

EXAMPLE 5

Carrying out the cyclic voltammetry measurements. The cyclic voltammetrymeasurements were made using a computer-controlled bipotentiostat (CHInstruments, Model 832) at room temperature in a standard cell having a3-electrode configuration. The modified gold electrode was used as theworking electrode, a platinum wire served as the auxiliary electrode(counter electrode), and an Ag/AgCl electrode with internal saturatedKCl solution, separated from the probe space via a Luggin capillary, wasused as the reference electrode to determine the potential. Serving asan electrolyte was 0.7 molar TEATFB or 1 molar NaCl. A cyclicvoltammogram of the Au—S-ds-oligo-PQQ electrode is shown in FIG. 4 incomparison with an Au—S-ss-oligo-PQQ electrode, and the effect of the 2bp mismatches on the cyclic voltammogram of the Au—S-ds-oligo-PQQelectrode is shown in FIG. 5. The potentials are each indicated as E-Eo,i.e. relative to the half-wave potential.

In FIG. 4, shows clearly a significantly greater current flow ascompared with the non-hybridized form is clearly evident when adouble-strand oligonucleotide is present. This allows sequence-specifichybridization events to be detected. From FIG. 5 it becomes clear that,in the case of hybridization with a target oligonucleotide strand thatexhibits 2 base-pair mismatches, for one thing, a weaker current flows,and for another, the difference of the current maximums is increased.

1. A method of producing a modified conductive surface, wherein anucleic acid oligomer or a nucleic acid oligomer modified by attaching aredox-active substance that is selectively oxidizable and reducible at apotential φ with 2.0 V≧φ≦−2.0 V, measured against a normal hydrogenelectrode, is hybridized with the nucleic acid oligomer strand which iscomplementary to the nucleic acid oligomer or the modified nucleic acidoligomer and applied to a conductive surface and is in the form of thedouble-strand hybrid; and one or more kinds of nucleic acid oligomers inthe form of the double-strand hybrid are bound to a conductive surfaceand only the nucleic acid oligomers bound to the conductive surface aremodified by attaching a redox-active substance to the nucleic acidoligomers.
 2. A method of producing a modified conductive surface,wherein a nucleic acid oligomer or a nucleic acid oligomer modified byattaching a redox-active substance that is selectively oxidizable andreducible at a potential φ with 2.0 V≧φ≧−2.0 V, measured against anormal hydrogen electrode, is hybridized with the nucleic acid oligomerstrand which Is complementary to the nucleic acid oligomer or themodified nucleic acid oligomer and applied to a conductive surface andis in the form of the double-strand hybrid, which is thermallydehybridized following application to the conductive surface.
 3. Themethod according to claim 2, wherein the double-strand hybrid is appliedto the conductive surface in the presence of further chemical compoundsalso attached to the conductive surface.
 4. The method according toclaim 2, wherein the nucleic acid oligomers or the modified nucleic acidoligomers are attached to the conductive surface covalently or by meansof physisorption.
 5. The method according to claim 2, wherein thenucleic acid oligomers or the modified nucleic add oligomers arecovalently attached to branched or linear molecular moieties of anycomposition and chain length and these molecular moieties are attachedto the conductive surface covalently or by mean so physisorption.
 6. Themethod according to claim 2, wherein one or more kinds of nucleic acidoligomers in the form of the double-strand hybrid are bound to aconductive surface and only the nucleic acid oligomers bound to theconductive surface are modified by attaching a redox-active substance tothe nucleic acid oligomers.